Toxoplasma gondii infections are a public health hazard for millions of individuals that contact this pathogen annually, particularly those with immune systems weakened by aging, chemotherapy or AIDS; and women that acquire the infection during pregnancy. Exposure to this pathogen occurs through contact with environmental sources and contaminated food ranking T. gondii only behind bacterial pathogens, Listeria and Salmonella, as the third-leading cause of food-borne death. No current therapy is able to prevent life- time infections caused by the encysted bradyzoite stage and drug toxicity limits long-term treatment for at risk individuals. Thus, there is a critical need to develop new approaches capable of preventing chronic Toxoplasma infection, although achieving this goal is hampered by a lack of understanding of the molecular basis for tissue cyst formation. Recently, we have completed a comprehensive SAGE analysis (funded by R21 award AI53815) of the transcriptome of the Toxoplasma intermediate life cycle. These studies demonstrate primary developmental transitions leading to tissue cyst formation are accompanied by a temporally ordered set of transcriptional events from genes that are dispersed across all parasite chromosomes, and are therefore, co-regulated by trans-acting protein factors. This exploratory grant seeks to gain a greater understanding of the core transcriptional mechanisms associated with parasite development by characterizing the nucleotide sequence elements and identifying the protein factors involved in the expression of known and novel bradyzoite-specific genes. We have developed dual- luciferase assays based on high-throughput cloning methods to functionally demonstrate that the control of tachyzoite- and bradyzoite-specific gene expression requires gene proximal sequences lying upstream of coding regions in the Toxoplasma genome. In this proposal, we will employ these same methods to define the minimal sequence requirements for five bradyzoite promoters, and in turn, use this information to identify the appropriate DNA binding proteins through biochemical and genetic strategies. The studies proposed here will address a fundamental gap in our understanding how Toxoplasma parasites (and other Apicomplexa) regulate gene expression, and will ultimately, lead to a better understanding of how these parasites control development into the clinically important tissue cyst. [unreadable] [unreadable] [unreadable]